Project Summary/Abstract Alterations in transcription factor signaling either through loss of function of tumor suppressors, such as p53, or by inappropriate activation of oncogenes, such as myc, is a common theme across all cancer types. Despite a vast amount of research, the underlying mechanisms by which many transcription factors contribute to tumor progression and maintenance remain unclear. The goal of this project is to identify novel transcription factor- associated proteins that regulate essential functions in cancer cells and have the potential to be targeted therapeutically. This proposal describes a multi-faceted approach to understanding the interplay between transcription factors and their associated proteins in the pathogenesis of Squamous Cell Carcinoma (SCC). SCC is associated with high morbidity and mortality, and is a particularly good model system for the study of transcription factor biology in cancer. SCC can present in many different epithelial tissues, but a common molecular feature is over-expression or genomic amplification of the p63 transcription factor, suggesting an important role in this tumor type. Importantly, normal epithelial cells have a different physiological and molecular response to p63 loss than SCC cells, due to the high expression of the pro-apoptotic protein p73. In SCC cells, p63 suppresses p73 activity through direct binding, and thus is essential for maintaining cell survival. In addition, p63 has been shown to regulate a variety of other essential pathways in normal epithelial cells, but the in vivo significance of each of these pathways in SCC is unclear. In order to address this issue, a mouse model of SCC has been generated in which p63 can be genetically inactivated specifically in tumor cells of established, invasive SCC. This murine model is particularly relevant to human cancer, as patients normally present clinically with detectable tumors, which requires treatment to slow tumor progression. Analysis of the physiological and molecular changes that accompany p63 loss in murine tumors has provided insight into the key role of p63 in the maintenance of SCC. Experiments contained in this proposal will address the mechanisms of p63-mediated survival that are amenable to therapeutic intervention. Importantly, this same model system can also be used to examine the role of other proteins thought to be important in the maintenance of SCC in future studies. While it is important to understand the key pathways controlled by p63 in vivo, development of effective therapies requires knowledge of how p63 regulates key target genes. This question will be investigated by biochemical identification of proteins that are directly associated with p63 using tandem affinity purification (TAP), which has already been successfully performed. Novel proteins will be evaluated for their ability to promote survival of SCC cells through the regulation of p63 transcriptional target genes. In particular, I will expand my current knowledge base through the use of whole-genome technologies to identify the common and unique p63-target genes regulated by different p63-interacting proteins. In addition, these systems can all be easily adapted to examine other transcription factors that contribute to the pathogenesis of SCC. These studies will contribute to the understanding of the key functions of over-expressed transcription factors in SCC, and validate their potential for therapeutic targeting by examining their role in a relevant in vivo model system. The mentored phase of this research project will be performed in the lab of Leif Ellisen at the Massachusetts General Hospital (MGH) Cancer Center. In the Ellisen laboratory, Dr. Ramsey has developed many new methodologies and model systems that can easily be adapted for use in an independent laboratory. These include the modification of TAP for use in SCC cell lines to identify novel transcription co-factors, and development of murine cancer models suitable for examining transcription factor biology in vivo. New training in bioinformatics and whole-genome analysis of transcription will complement these skills, and provide a solid foundation of technical skills required to work with the vast new in silico data being produced. These model systems provide a strong foundation to build towards the long-term goal of developing a research program that combines biochemical characterization of transcription complexes with in vivo validation of these complexes in relevant murine cancer models. The mentored phase of this award will allow Dr. Ramsey to continue to acquire new technical skills and reagents necessary for running a successful research program, while taking advantage of the many resources available in the Ellisen lab. These include access to technologies and core facilities needed to complete the experiments in this proposal, close collaboration with members of the MGH Cancer Center, and frequent opportunities to present data to the local and international scientific community. In addition, the MGH Cancer Center and Harvard Medical School community offer many career development resources specifically aimed at post-doctoral fellows to facilitate the transition to an independent investigator, such as networking and interviewing seminars. These many resources will help to ensure the successful completion of the proposed experiments, and transition of Dr. Ramsey from post-doctoral fellow to independent academic researcher.